Rna-interference by single-stranded rna molecules

ABSTRACT

The present invention relates to sequence and structural features of single-stranded (ss)RNA molecules required to mediate target-specific nucleic acid modifications by RNA-interference (RNAi), such as target mRNA degradation and/or DNA methylation.

This application is a divisional of U.S. Ser. No. 10/520,470 filed Jan.7, 2005, which is a 35 U.S.C. 371 National Phase Entry Application fromPCT/EP2003/007516, filed Jul. 10, 2003, which claims the benefit ofEuropean Patent Application Nos. 02015532.1 filed Jul. 10, 2002 and02018906.4 filed Aug. 23, 2002, the disclosures of which is incorporatedherein in their entirety by reference.

DESCRIPTION

The present invention relates to sequence and structural featUres ofsingle-stranded (ss)RNA molecules required to mediate target-specificnucleic acid modifications by RNA-interference (RNAi), such as targetmRNA degradation and/or DNA methylation.

Most eukaryotes possess a cellular defense system protecting theirgenomes against invading foreign genetic elements. Insertion of foreignelements is believed to be generally accompanied by formation of dsRNAthat is interpreted by the cell as a signal for unwanted gene activity(e.g. Ahlquist, Science 296 (2002), 1270-1273; Fire et al., Nature 391(1998), 806-811). Dicer RNase III rapidly processes dsRNA to small dsRNAfragments of distinct size and structure (e.g. Bernstein et al., Nature409 (2001), 363-366), the small interfering RNAs (siRNAs) (Elbashir etal., Genes & Dev. 15 (2001 b), 188-200), which direct thesequence-specific degradation of the single-stranded mRNAs of theinvading genes. siRNA duplexes have 2- to 3-nt 3′ overhanging ends andcontain 5′ phosphate and free 3′ hydroxyl termini (WO 02/44321). Theprocess of posttranscriptional dsRNA-dependent gene silencing iscommonly referred to as RNA interference (RNAi), and in some instancesis also linked to transcriptional silencing.

Experimental introduction of siRNA duplexes into mammalian cells is nowwidely used to disrupt the activity of cellular genes homologous insequence to the introduced dsRNA. Used as a reverse genetic approach,siRNA-induced gene silencing accelerates linking of gene sequence tobiological function. siRNA duplexes are short enough to bypass generaldsRNA-induced unspecific effects in vertebrate animal and mammaliancells. siRNAs may also be expressed intracellularly from introducedexpression plasmids or viral vectors providing an alternative tochemical RNA synthesis. Therefore, an understanding of how siRNAs act inmammalian systems is important for refining this gene silencingtechnology and for producing gene-specific therapeutic agents.

Biochemical studies have begun to unravel the mechanistic details ofRNAi. The first cell-free systems were developed using D. melanogastercell or embryo extracts, and were followed by the development of invitro systems from C. elegans embryo and mouse embryonal carcinomacells. While the D. melanogaster lysates support the steps of dsRNAprocessing and sequence-specific mRNA targeting, the latter two systemsonly recapitulate the first step.

RNAi in D. melanogaster extracts is initiated by ATP-dependentprocessing of long dsRNA to siRNAs by Dicer RNase III (e.g. Bernstein etal., (2001), supra). Thereafter, siRNA duplexes are assembled into amulti-component complex, which guides the sequence-specific recognitionof the target mRNA and catalyzes its cleavage (e.g. Elbashir (2001 b),supra). This complex is referred to as RNA-induced silencing complex(RISC) (Hammond et at., Nature 404 (2000), 293-296). siRNAs in D.melanogaster are predominantly 21- and 22-nt, and when paired in amanner to contain a 2-nt 3′ overhanging structure effectively enter RISC(Elbashir et al., EMBO J. 20 (2001 c), 6877-6888). Mammalian systemshave siRNAs of similar size, and siRNAs of 21- and 22-nt also representthe most effective sizes for silencing genes expressed in mammaliancells (e.g. Elbashir et al., Nature 411 (2001 a), 494-498, Elbashir etal., Methods 26 (2002), 199-213).

RISC assembled on siRNA duplexes in D. melanogaster embryo lysatetargets homologous sense as well as antisense single-stranded RNAs fordegradation. The cleavage sites for sense and antisense target RNAs arelocated in the middle of the region spanned by the siRNA duplex.Importantly, the 5′-end, and not the 3′ :end, of the guide siRNA setsthe ruler for the position of the target RNA cleavage. Furthermore, a 5′phosphate is required at the target-complementary strand of a siRNAduplex for RISC activity, and ATP is used to maintain the 5′ phosphatesof the siRNAs (Nykänen et al., Cell 107 (2001), 309-321). SyntheticsiRNA duplexes with free 5′ hydroxyls and 2-nt 3′ overhangs are soreadily phosphorylated in D. melanogaster embryo lysate that the RNAiefficiencies of 5′-phosphorylated and non-phosphorylated siRNAs are notsignificantly different (Elbashir et al. (2001 c), supra).

Unwinding of the siRNA duplex must occur prior to target RNArecognition. Analysis of ATP requirements revealed that the formation ofRISC on siRNA duplexes required ATP in lysates of D. melanogaster. Onceformed, RISC cleaves the target RNA in the absence of ATP. The need forATP probably reflects the unwinding step and/or other conformationalrearrangements. However, it is currently unknown if the unwound strandsof an siRNA duplex remain associated with RISC or whether RISC onlycontains a single-stranded siRNA.

A component associated with RISC was identified as Argonaute2 from D.melanogaster Schneider 2 (S2) cells (Hammond et al., Science 293 (2001a), 1146-1150), and is a member of a large family of proteins. Thefamily is referred to as Argonaute or PPD family and is characterized bythe presence of a PAZ domain and a C-terminal Piwi domain, both ofunknown function (Cerutti et al., Trends Biochem. Sci. (2000), 481-482);Schwarz and Zamore, Genes & Dev. 16 (2002), 1025-1031). The PAZ domainis also found in Dicer. Because Dicer and Argonaute2 interact in S2cells, PAZ may function as a protein-protein interaction motif.Possibly, the interaction between Dicer and Argonaute2 facilitates siRNAincorporation into RISC. In D. melanogaster, the Argonaute family hasfive members, most of which were shown to be involved in gene silencingand development. The mammalian members of the Argonaute family arepoorly characterized, and some of them have been implicated intranslational control, microRNA processing and development. Thebiochemical function of Argonaute proteins remains to be established andthe development of more biochemical systems is crucial.

Here we report on the analysis of human RISC in extracts prepared fromHeLa cells. The reconstitution of RISC and the mRNA targeting steprevealed that RISC is a ribonucleoprotein complex that is composed of asingle-stranded siRNA. Once RISC is formed the incorporated siRNA can nolonger exchange with free siRNAs. Surprisingly, RISC can bereconstituted in HeLa S100 extracts providing single-stranded siRNAs.Introducing 5′ phosphorylated single-stranded antisense siRNAs into HeLacells potently silences an endogenous gene with similar efficiency thanduplex siRNA.

The object underlying the present invention is to provide novel agentscapable of mediating target-specific RNAi.

The solution of this problems is provided by the use of asingle-stranded RNA molecule for the manufacture of an agent forinhibiting the expression of said target transcript. Surprisingly, itwas found that single-stranded RNA molecules are capable of inhibitingthe expression of target transcripts by RNA-interference (RNAi).

The length of the single-stranded RNA molecules is preferably from 14-50nt, wherein at least the 14 to 20 5′-most nucleotides are substantiallycomplementary to the target RNA transcript. The RNA oligonucleotides mayhave a free 5′ hydroxyl moiety, or a moiety which is 5′ phosphorylated(by means of chemical synthesis or enzymatic reactions) or which ismodified by 5′-monophosphate analogues.

The inhibition of target transcript expression may occur in vitro, e.g.in eucaryotic, particularly mammalian cell cultures or cell extracts. Onthe other hand, the inhibition may also occur in vivo i.e. ineucaryotic, particularly mammalian organisms including human beings.

Preferably, the single-stranded RNA molecule has a length from 15-29nucleotides. The RNA-strand may have a 3′ hydroxyl group. In some cases,however, it may be preferable to modify the 3′ end to make it resistantagainst 3′ to 5′ exonucleases. Tolerated 3′-modifications are forexample terminal 2′-deoxy nucleotides, 3′ phosphate, 2′,3′-cyclicphosphate, C3 (or C6, C7, C12) aminolinker, thiol linkers, carboxyllinkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethyleneglycol, hexaethylene glycol), biotin, fluoresceine, etc.

The 5′-terminus comprises an OH group, a phosphate group or an analoguethereof. Preferred 5′ phosphate modifications are 5′-monophosphate((HO)₂(O)P—O-5′), 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′),5′-triphosphate ((HO)₂(O) P—O—(HO)(O)P—O—P(HO) (O)—O-5′), 5′-guanosinecap (7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine cap.(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-monothiophosphate(phosphorothioate; (HO)₂(S)P—O-5′), 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)₂(O)P—S-5′); any additional combination of oxgen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

The sequence of the RNA molecule of the present invention has to have asufficient identity to a nucleic acid target molecule in order tomediate target-specific RNAi. Thus the single-stranded RNA molecule ofthe present invention is substantially complementary to the targettranscript.

The target RNA cleavage reaction guided by the single-stranded RNAmolecules of the present invention is highly sequence-specific. However,no all positions of the RNA molecule contribute equally to targetrecognition. Mismatches, particularly at the 3′-terminus of thesingle-stranded RNA molecule, more particularly the residues 3′ to thefirst 20 nt of the single-stranded RNA molecule are tolerated.Especially preferred are single-stranded RNA molecules having at the5′-terminus at least 15 and preferably at least 20 nucleotides which arecompletely complementary to a predetermined target transcript or have atonly mismatch and optionally up to 35 nucleotides at the 3′-terminuswhich may contain 1 or several, e.g. 2, 3 or more mismatches.

In order to enhance the stability of the single-stranded RNA molecules,the 3′-ends may be stabilized against degradation, e.g. they may beselected such that they consist of purine nucleotides, particularlyadenosine or guanosine nucleotides. Alternatively or additionally, 3′nucleotides may be substituted by modified nucleotide analogues,including backbone modifications of ribose and/or phosphate residues.

In an especially preferred embodiment of the present invention the RNAmolecule may contain at least one modified nucleotide analogue. Thenucleotide analogues may be located at positions where thetarget-specific activity, e.g. the RNAi mediating activity is notsubstantially affected, e.g. in a region at the 5′-end and/or the 3′-endof the RNA molecule. Particularly, the 3′-terminus may be stabilized byincorporating modified nucleotide analogues, such as non-nucleotidicchemical derivatives such as C3 (or C6, C7, C12) arninolinker, thiollinkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12,abasic, triethylene glycol, hexaethylene glycol), biotin, fluoresceine,etc. A further modification, by which the nuclease resistance of the RNAmolecule may be increased, is by covalent coupling of invertednucleotides, e.g. 2′-deoxyribonucleotides or ribonucleotides to the3′-end of the RNA molecule. A preferred RNA molecule structurecomprises: 5′-single-stranded siRNA-3′-O—P(O)(OH)—O-3′-N, wherein N is anucleotide, e.g. a 2′-deoxyribonucleotide or ribonucleotide, typicallyan inverted thymidine residue, or an inverted oligonucleotide structure,e.g. containing up to 5 nucleotides.

Preferred nucleotide analogues are selected from sugar- orbackbone-modified ribonucleotides. It should be noted, however, thatalso nucleobase-modified ribonucleotides, i.e. ribonucleotides,containing a non-naturally occurring nucleobase instead of a naturallyoccurring nucleobase such as uridines or cytidines modified at the5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine;5-methyl-cytidine; adenosines and guahosines modified at the 8-position,e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable. Inpreferred sugar-modified ribonucleotides the 2′ OH-group is replaced bya group selected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or CN,wherein R is C₁-C₆ alkyl, alkenyl, alkynyl or methoxyethoxy, and halo isF, Cl, Br or I. In preferred backbone-modified ribonucleotides thephosphoester group connecting to adjacent ribonucleotides is replaced bya modified group, e.g. a phosphorothioate, phosphorodithioate, N3′-O5′-and/or N5′-O3′ phosphoramidate group. It should be noted that the abovemodifications may be combined. For example, complementary ornon-complementary nucleotides at the 3′-terminus, particularly after atleast 15, more particularly after at least 20 5′-terminal nucleotidesmay be modified without significant loss of activity.

The single-stranded RNA molecule of the invention may be prepared bychemical synthesis. Methods of synthesizing RNA molecules are known inthe art.

The single-stranded RNAs can also be prepared by enzymatic transcriptionfrom synthetic DNA templates or from DNA plasmids isolated fromrecombinant bacteria and subsequent 5′-terminal modification. Typically,phage RNA polymerases are used such as T7, T3 or SP6 RNA polymerase.

A further aspect of the present invention relates to a method ofmediating RNA interference in a cell or an organism comprising thesteps:

-   -   (a) contacting the cell or organism with the single-stranded RNA        molecule of the invention under conditions wherein        target-specific nucleic acid modifications may occur and    -   (b) mediating a target-specific nucleic acid modification        effected by the single-stranded RNA towards a target nucleic        acid having a sequence portion substantially complementary to        the single-stranded RNA.

Preferably the contacting step (a) comprises introducing thesingle-stranded RNA molecule into a target cell, e.g. an isolated targetcell, e.g. in cell culture, a unicellular microorganism or a target cellor a plurality of target cells within a multicellular organism. Morepreferably, the introducing step comprises a carrier-mediated delivery,e.g. by liposomal carriers and/or by injection. Further suitabledelivery systems include Oligofectamine (Invitrogen) and Transit-TKOsiRNA Transfection reagent (Mirus)

The method of the invention may be used for determining the function ofa gene in a cell or an organism or even for modulating the function of agene in a cell or an organism, being capable of mediating RNAinterference.

The cell is preferably a eukaryotic cell or a cell line, e.g. a plantcell or an animal cell, such as a mammalian cell, e.g. an embryoniccell, a pluripotent stem cell, a tumor cell, e.g. a teratocarcinoma cellor a virus-infected cell. The organism is preferably a eukaryoticorganism, e.g. a plant or an animal, such as a mammal, particularly ahuman.

The target gene to which the RNA molecule of the invention is directedmay be associated with a pathological condition. For example, the genemay be a pathogen-associated gene, e.g. a viral gene, a tumor-associatedgene or an autoimmune disease-associated gene. The target gene may alsobe a heterologous gene expressed in a recombinant cell or a geneticallyaltered organism. By determinating or modulating, particularly,inhibiting the function of such a gene valuable information andtherapeutic benefits in the agricultural field or in the medicine orveterinary medicine field may be obtained.

The ssRNA is usually administered as a pharmaceutical composition. Theadministration may be carried out by known methods, wherein a nucleicacid is introduced into a desired target cell in vitro or in vivo.Commonly used gene transfer techniques include calcium phosphate,DEAE-dextran, electroporation and microinjection and viral methods(Graham, F. L. and van der Eb, A. J. (1973) Virol. 52, 456; McCutchan,J. H. and Pagano, J. S. (1968), J. Natl. Cancer Inst. 41, 351; Chu, G.et al (1987), Nucl. Acids Res. 15, 1311; Fraley, R. et al. (1980), J.Biol. Chem. 255, 10431; Capecchi, M. R. (1980), Cell 22, 479). A recentaddition to this arsenal of techniques for the introduction of nucleicacids into cells is the use of cationic liposomes (Feigner, P. L. et al.(1987), Proc. Natl. Acad. Sci USA 84, 7413). Commercially availablecationic lipid formulations are e.g. Tfx 50 (Promega) orLipofectamin2000 (Life Technologies). A further preferred method for theintroduction of RNA into a target organism, particularly into a mouse,is the high-pressure tail vein injection (Lewis, D. L. et al. (2002),Nat. Genet.29, 29; McCaffrey, A. P. et al. (2002), Nature 418, 38-39).

Herein, a buffered solution comprising the single-stranded RNA (e.g.about 2 ml) is injected into the tail vein of the mouse within 10 s.

Thus, the invention also relates to a pharmaceutical compositioncontaining as an active agent at least one single-stranded RNA moleculeas described above and a pharmaceutical carrier. The composition may beused for diagnostic and for therapeutic applications in human medicineor in veterinary medicine.

For diagnostic or therapeutic applications, the composition may be inform of a solution, e.g. an injectable solution, a cream, ointment,tablet, suspension or the like. The composition may be administered inany suitable way, e.g. by injection, by oral, topical, nasal, rectalapplication etc. The carrier may be any suitable pharmaceutical carrier.Preferably, a carrier is used, which is capable of increasing theefficacy of the RNA molecules to enter the target-cells. Suitableexamples of such carriers are liposomes, particularly cationicliposomes. A further preferred administration method is injection.

A further preferred application of the RNAi method is a functionalanalysis of eukaryotic cells, or eukaryotic non-human organisms,preferably mammalian cells or organisms and most preferably human cells,e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. Bytransfection with suitable single-stranded RNA molecules which arehomologous to a predetermined target gene or DNA molecules encoding asuitable single-stranded RNA molecule a specific knockout phenotype canbe obtained in a target cell, e.g. in cell culture or in a targetorganism. The presence of short single-stranded RNA molecules does notresult in an interferon response from the host cell or host organism.

In an especially preferred embodiment, the RNA molecule is administeredassociated with biodegradable polymers, e.g. polypeptides,poly(d,l-lactic-co-glycolic acid) (PLGA), polylysine or polylysineconjugates, e.g. polylysine-graft-imidazole acetic acid, orpoly(beta-amino ester) or microparticles, such as microspheres,nanoparticles or nanospheres. More preferably the RNA molecule iscovalently coupled to the polymer or microparticle, wherein the covalentcoupling particularly is effected via the 3′-terminus of the RNAmolecule.

Further, the invention relates to a pharmaceutical composition forinhibiting the expression of a target transcript by RNAi comprising asan active agent a single-stranded RNA molecule having a length from14-50, preferably 15-29 nucleotides wherein at least the 14-20 5′mostnucleotides are substantially complementary to said target transcript.

Furthermore, the invention relates to a method for the prevention ortreatment of a disease associated with overexpression of at least onetarget gene comprising administering a subject in need thereof asingle-stranded RNA molecule having a length from 14-50, preferably15-29 nucleotides wherein at least the 14-20 5′most nucleotides aresubstantially complementary to a target transcript in an amount which istherapeutically effective for RNAi.

Still, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout phenotype comprising an at least partially deficient expressionof at least one endogeneous target gene wherein said cell or organism istransfected with at least one single-stranded RNA molecule capable ofinhibiting the expression of at least one endogeneous target gene. Itshould be noted that the present invention allows the simultaneousdelivery of several antisense RNAs of different sequences, which areeither cognate to a different or the same target gene.

Gene-specific knockout phenotypes of cells or non-human organisms,particularly of human cells or non-human mammals may be used in analyticprocedures, e.g. in the functional and/or phenotypical analysis ofcomplex physiological processes such as analysis of gene expressionprofiles and/or proteomes. For example, one may prepare the knock-outphenotypes of human genes in cultured cells which are assumed to beregulators of alternative splicing processes. Among these genes areparticularly the members of the SR splicing factor family, e.g. ASF/SF2,SC35, SRp2O, SRp4O or SRp55. Further, the effect of SR proteins on themRNA profiles of predetermined alternatively spliced genes such as CD44may be analysed. Preferably the analysis is carried out byhigh-throughput methods using oligonucleotide based chips.

Using RNAi based knockout technologies, the expression of an endogeneoustarget gene may be inhibited in a target cell or a target organism. Theendogeneous gene may be complemented by an exogeneous target nucleicacid coding for the target protein or a variant or mutated form of thetarget protein, e.g. a gene or a cDNA, which may optionally be fused toa further nucleic acid sequence encoding a detectable peptide orpolypeptide, e.g. an affinity tag, particularly a multiple affinity tag.Variants or mutated forms of the target gene differ from the endogeneoustarget gene in that they encode a gene product which differs from theendogeneous gene product on the amino acid level by substitutions,insertions and/or deletions of single or multiple amino acids. Thevariants or mutated forms may have the same biological activity as theendogeneous target gene. On the other hand, the variant or mutatedtarget gene may also have a biological activity, which differs from thebiological activity of the endogeneous target gene, e.g. a partiallydeleted activity, a completely deleted activity, an enhanced activityetc.

The complementation may be accomplished by coexpressing the polypeptideencoded by the exogeneous nucleic acid, e.g. a fusion protein comprisingthe target protein and the affinity tag and the double stranded RNAmolecule for knocking out the endogeneous gene in the target cell. Thiscoexpression may be accomplished by using a suitable expression vectorexpressing both the polypeptide encoded by the exogeneous nucleic acid,e.g. the tag-modified target protein and the single-stranded RNAmolecule or alternatively by using a combination of expression vectors.Proteins and protein complexes which are synthesized de novo in thetarget cell will contain the exogeneous gene product, e.g. the modifiedfusion protein. In order to avoid suppression of the exogeneous geneproduct expression by the RNAi molecule, the nucleotide sequenceencoding the exogeneous nucleic acid may be altered on the DNA level(with or without causing mutations on the amino acid level) in the partof the sequence which is homologous to the single-stranded RNA molecule.Alternatively, the endogeneous target gene may be complemented bycorresponding nucleotide sequences from other species, e.g. from mouse.

Preferred applications for the cell or organism of the invention is theanalysis of gene expression profiles and/or proteomes. In an especiallypreferred embodiment an analysis of a variant or mutant form of one orseveral target proteins is carried out, wherein said variant or mutantforms are reintroduced into the cell or organism by an exogeneous targetnucleic acid as described above. The combination of knockout of anendogeneous gene and rescue, by using mutated, e.g. partially deletedexogeneous target has advantages compared to the use. of a knockoutcell. Further, this method is particularly suitable for identifyingfunctional domains of the target protein. In a further preferredembodiment a comparison, e.g. of gene expression profiles and/orproteomes and/or phenotypic characteristics of at least two cells ororganisms is carried out. These organisms are selected from:

-   -   (i) a control cell or control organism without target gene        inhibition,    -   (ii) a cell or organism with target gene inhibition and    -   (iii) a cell or organism with target gene inhibition plus target        gene complementation by an exogeneous target nucleic acid.

The method and cell of the invention may also be used in a procedure foridentifying and/or characterizing pharmacological agents, e.g.identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents.

Thus, the present invention also relates to a system for identifyingand/or characterizing pharmacological agents acting on at least onetarget protein comprising:

-   -   (a) a eukaryotic cell or a eukaryotic non-human organism capable        of expressing at least one endogeneous target gene coding for        said target protein,    -   (b) at least one single-stranded RNA molecule capable of        inhibiting the expression of said at least one endogeneous        target gene by RNAi and    -   (c) a test substance or a collection of test substances wherein        pharmacological properties of said test substance or said        collection are to be identified and/or characterized.

Further, the system as described above preferably comprises:

-   -   (d) at least one exogeneous target nucleic acid coding for the        target protein or a variant or mutated form of the target        protein wherein said exogeneous target nucleic acid differs from        the endogeneous target gene on the nucleic acid level such that        the expression of the exogeneous target nucleic acid is        substantially less inhibited by the single-stranded RNA molecule        than the expression of the endogeneons target gene.

Furthermore, the RNA knockout complementation method may be used forpreparative purposes, e.g. for the affinity purification of proteins orprotein complexes from eukaryotic cells, particularly mammalian cellsand more particularly human cells. In this embodiment of the invention,the exogeneous target nucleic acid preferably codes for a target proteinwhich is fused to an affinity tag.

The preparative method may be employed for the purification of highmolecular weight protein complexes which preferably have a mass of ≧150kD and more preferably of >500 kD and which optionally may containnucleic acids such as RNA. Specific examples are the heterotrimericprotein complex consisting of the 20 kD, 60 kD and 90 kD proteins of theU4/U6 snRNP particle, the splicing factor SF3b from the 17S U2 snRNPconsisting of 5 proteins having molecular weights of 14, 49, 120, 145and 155 kD and the 25S U4/U6/U5 tri-snRNP particle containing the U4, U5and U6 snRNA molecules and about 30 proteins, which has a molecularweight of about 1.7 MD.

This method is suitable for functional proteome analysis in mammaliancells, particularly human cells.

Finally, the invention relates to a purified and isolated mammalian,partiCularly human RNA-induced silencing complex (RISC) having anapparent molecular weight of less than about 150-160 kDa, e.g. about 120to 150-160 kDa. The RISC comprises polypeptide and optionally nucleicacid components, particularly single-stranded RNA molecules as describedabove. The RISC may be used as a target for diagnosis and/or therapy, asa diagnostic and/or therapeutic agent itself, as a molecular-biologicalreagent or as component in a screening procedure for the identificationand/or characterization of pharmaceutical agents.

Polypeptide components of RISC preferably comprise members of theArgonaute family of proteins, and contain eIF2C1 and/or eIF2C2, andpossibly at least one other expressed eIF2C family member, particularlyselected from eIF2C3, eIF2C4, HILI and HIWI.

Expression or overexpression of one or several proteins present in RISCin suitable host cells, e.g. eukaryotic cells, particularly mammaliancells, is useful to assist an RNAi response. These proteins may also beexpressed or overexpressed in transgenic animals, e.g. vertebrates,particularly mammals, to produce animals particularly sensitive toinjected single-stranded or double-stranded siRNAs. Further, the genesencoding the proteins may be administered for therapeutic purposes, e.g.by viral or non-viral gene delivery vectors.

It is also conceivable to administer a siRNA/eIF2C1 or 2 complexdirectly by the assistance of protein transfection reagents (e.g.Amphoteric Protein Transfection Reagents, ProVectin protein (lmgenex),or similar products) rather than RNA/DNA transfection. This may havetechnical advantages over siRNA transfection that are limited to nucleicacid transfection.

Alternatively to the application of siRNAs as synthetic double-strandedor single-stranded siRNAs, it is conceivable to also administer anantisense siRNA precursor molecule in the form of a hairpin stem-loopstructure comprising 19 to 29 base pairs in the stem with or without 5′or 3′ overhanging ends on one side of the duplex and a nucleotide ornon-nucleotide loop on the other end. Preferably, the hairpin structurehas a 3′ overhang of from 1-5 nucleotides. Further, the precursor maycontain modified nucleotides as described above, particularly in theloop and/or in the 3′ portion, particularly in the overhang. The siRNAor precursors of siRNAs may also be introduced by viral vectors or RNAexpression systems into a RISC compound, e.g. eIF2C1 and/or 2overexpressing organism or cell line. The siRNA precursors may also begenerated by direct expression within an organism or cell line. This maybe achieved by transformation with a suitable expression vector carryinga nucleic acid template operatively linked to an expression controlsequence to express the siRNA precursor.

Further, the present invention is explained in more detail in thefollowing figures and examples.

FIGURE LEGENDS

FIG. 1. HeLa cytoplasmic S100 extracts show siRNA-dependent target RNAcleavage.

(A) Representation of the 177-nt ³²P-cap-labeled target RNA with thetargeting siRNA duplex. Target RNA cleavage site and the length of theexpected cleavage products is also shown. The fat black line positionedunder the antisense siRNA is used in the following figures as symbol toindicate the region of the target RNA, which is complementary to theantisense siRNA sequence. (B) Comparison of the siRNA mediated targetRNA cleavage using the previously established D. melanogaster embryo invitro system and HeLa cell S100 cytoplasmic extract. 10 nM cap-labeledtarget RNA was incubated with 100 nM siRNA as described in materials.Reaction products were resolved on a 6% sequencing gel. Position markerswere generated by partial RNase T1 digestion (T1) and partial alkalinehydrolysis (OH) of the cap-labeled target RNA. The arrow indicates the5′ cleavage product, the fragment is unlabeled and therefore invisible.

FIG. 2. Chemical modification of the 5′ end of the antisense but not thesense siRNAs prevents sense target RNA cleavage in HeLa S100 extracts.(A) Illustration of the possible 5′ and 3′ aminolinker modifications ofthe sense and antisense strands of a siRNA duplex. L5 represents a6-carbon chain aminolinker connected via a 5′-phosphodiester linkage, L3represents a 7-carbon aminolinker connected via a phosphodiester bond tothe terminal 3′ phosphate. s, sense; as, antisense. (B) Target RNAcleavage testing various combinations of 5′ and 3′ aminolinker-modifiedsiRNA duplexes. NC (negative control) shows an incubation reaction ofthe target RNA in the absence of siRNA duplex. T1, RNase T1 ladder; OH,partial alkaline hydrolysis ladder.

FIG. 3. siRNA containing 3′-terminal phosphates are subjected toligation as well as dephosphorylation reactions.

(A) Sequence of the radiolabeled siRNA duplex. The labeled nucleotidewas joined to synthetic 20-nt antisense siRNA by T4 RNA ligation of³²pCp. The various combinations of 5′ and 3′ hydroxyl/phosphate wereprepared as described in materials. X and Y indicate 5′ and 3′modifications of the antisense siRNA. (B) Fate of the antisense siRNAduring incubation of the modified siRNA duplexes in HeLa S100 extract inthe presence of non-radiolabeled target RNA. The differentphosphorylated forms of the antisense siRNA were distinguished based ontheir gel mobility. Identical results were obtained when using 5′phosphorylated sense siRNA or when leaving out the target RNA duringincubation. Ligation products are only observed when 3′ phosphates werepresent on the labeled antisense siRNA.

FIG. 4: RISC is a stable complex that does not rapidly exchange boundsiRNA.

Increasing concentrations of non-specific siRNA compete withtarget-specific RISC formation when added simultaneously to HeLa S100extracts (lanes 4 to 7). However, when the unspecific siRNA duplex isadded 15 min after pre-incubation with the specific siRNA duplex, nomore competition was observed (3 lanes to the right). T1, RNase T1ladder.

FIG. 5. Partial purification of human RISC.

-   (A) Graphical representation of the structure of the biotinylated    siRNA duplex used for affinity purification of siRNA-associated    factors. L3 indicates a C7-aminolinker that was conjugated to a    photo-cleavable biotin N-hydroxysuccinimidyl ester; UV indicates    photocleavage of the UV-sensitive linkage to release affinity    selected complexes under native conditions. (B) Superdex-200 gel    filtration analysis of siRNA-protein complexes (siRNPs) recovered by    UV treatment/elution (UV elu) from the streptavidin affinity column.    Fractions were assayed for their ability to sequence-specifically    cleave the cap-labeled target RNA. The number of the 10 collected    fractions and the relative positions of the aldolase (158 kDa) and    BSA (66 kDa) size markers are indicated. (C) Glycerol gradient    (5%-20%) sedimentation of siRNPs recovered by UV treatment/elution    from the streptavidin affinity column. For legend, see (B). When    monitoring the precise size of target RNA cleavage fragments using    internally ³²P-UTP-labeled, capped mRNA, the sum is equal to the    full-length transcript, thus indicating that target RNA is indeed    only cleaved once in the middle of the region spanned by the siRNA.

FIG. 6. RISC contains a single-stranded siRNA.

siRNPs were subjected to affinity selection after incubation using siRNAduplexes with one or both strands biotinylated. The eluate recoveredafter UV treatment or the unbound fraction after streptavidin affinityselection (flow-through) was assayed for target RNA degradation. If theantisense strand was biotinylated, all sense target RNA-cleaving RISCwas bound to the streptavidin beads, while sense siRNA biotinylationresulted in RISC activity of the flow-through. The cleavage reaction inthe flow-through fraction was less efficient than in the UV eluate,because affinity-selected RISC was more concentrated.

FIG. 7. Single-stranded antisense siRNAs reconstitute RISC in HeLa S100extracts.

Analysis of RISC reconstitution using single-stranded or duplex siRNAscomparing HeLa S100 extracts (A) and the previously described D.melanogaster embryo lysate (B). Different concentrations ofsingle-stranded siRNAs (s, sense; as, antisense) and duplex siRNA (ds)were tested for specific targeting of cap-labeled substrate RNA. 100 nMconcentrations of the antisense siRNA reconstituted RISC in HeLa S100extract, although at reduced levels in comparison to the duplex siRNA.Reconstitution with single-stranded siRNAs was almost undetectable in D.melanogaster lysate, presumably because of the higher nuclease activityin this lysate causing rapid degradation of uncapped single-strandedRNAs.

FIG. 8. Single-stranded antisense siRNAs mediate gene silencing in HeLacells.

-   (A) Silencing of nuclear envelope protein lamin A/C. Fluorescence    staining of cells transfected with lamin A/C-specific siRNAs and GL2    luciferase (control) siRNAs. Top row, staining with lamin A/C    specific antibody; middle row, Hoechst staining of nuclear    chromatin; bottom row, phase contrast images of fixed cells. (B)    Quantification of lamin A/C knockdown after Western blot analysis.    The blot was stripped after lamin A/C probing and reprobed with    vimentin antibody. Quantification was performed using a Lumi-Imager    (Roche) and LumiAnalyst software to quantitate the ECL signals    (Amersham Biosciences), differences in gel loading were corrected    relative to non-targeted vimentin protein levels. The levels of    lamin A/C protein were normalized to the non-specific GL2 siRNA    duplex.

FIG. 9. Antisense siRNAs of different length direct target RNA cleavagein HeLa S100 extracts.

-   (A) Graphical representation of the experiment. Antisense siRNAs    were extended towards the 5′ side (series 1, 20 to 25-nt) or the 3′    side (series 2, 20 to 23-nt).-   (B) Target RNA cleavage using the antisense siRNAs described in (A).    HeLa S100 extract was incubated with 10 nM cap-labeled target RNA    and 100 nM antisense siRNAs at 30° C. for 2.5 h. Reaction products    were resolved on a 6% sequencing gel. Position markers were    generated by partial RNase T1 digestion (T1) and partial alkaline    hydrolysis (OH) of the cap-labeled target RNA. Arrows indicate the    position of the 5′ cleavage products generated by the different    antisense siRNAs. The fat black lines on the left (series 1) and the    right (series 2) indicate the region of the target RNA, which is    complementary to the antisense siRNA sequences.

FIG. 10. Length dependence of antisense siRNAs and effect of terminalmodifications for targeting RNA cleavage in HeLa S100 extracts.

HeLa S100 extract was incubated with 10 nM cap-labeled target RNA and100 nM antisense siRNAs at 30° C. for 2.5h. Reaction products wereresolved on a 6% sequencing get. Position markers were generated bypartial RNase T1 digestion (T1) of the cap-labeled target RNA. The fatblack line on the left indicates the region of the target RNA, which iscomplementary to the 21-nt antisense siRNA sequence. The siRNA sequencesused in each experiment are listed below (sense and antisense siRNAs arelisted together, they were pre-annealed to form duplex siRNAs). p,phosphate; t, 2′-deoxythymidine, c, 2′-deoxycytidine, g,2′-deoxycytidine, g, 2′-deoxyguanosine; L, aminolinker, B,photocleavable biotin; A,C,G,U, ribonucleotides.

Lane Sense siRNA (5′-3′) Antisense siRNA (5′-3′) 1 pUCGAAGUAUUCCG CG 2pUCGAAGUAUUCCG CGUACGUG 3 pUCGAAGUAUUCCG CGUACGUGAUGU 4 pUCGAAGUAUUCCGCGUACGUGAUGUUC 5 pUCGAAGUAUUCCG CGUACGUGAUGUUC AC 6 pUCGAAGUAUUCCG CG 7pUCGAAGUAUUCCG CGUACGUG 8 pUCGAAGUAUUCCG CGUACGUGAUGU 9 pUCGAAGUAUUCCGCGUACGUGAUGUUC 10 pUCGAAGUAUUCCG CGUACGUGAUGUUC AC 11 pUCGAAGUAUUCCGCGUACGUG 12 pUCGAAGUAUUCCG CGUACGtg 13 pUCGAAGUAUUCCG CGUACGUU 14pUCGAAGUAUUCCG CGUACGtt 15 pUCGAAGUAUUCCG CGUACGUG 16 pUCGAAGUAUUCCGCGUACGtg 17 pUCGAAGUAUUCCG CGUACGUU 18 pUCGAAGUAUUCCG CGUACGtt 19CGUACGCGGAAUACUUCG pUCGAAGUAUUCCG AAA CGUACGUG 20 CGUACGCGGAAUACUUCGpUCGAAGUAUUCCG AAA CGUACGtg 21 CGUACGCGGAAUACUUCG pUCGAAGUAUUCCG AAACGUACGUU 22 CGUACGCGGAAUACUUCG pUCGAAGUAUUCCG AAA CGUACGtt 23tCGAAGUAUUCCGC GUACGUULB 24 cGUACGCGGAAUACUUCG tCGAAGUAUUCCGC AUULBGUACGUULB 25 ptCGAAGUAUUCCGC GUACGttLB 26 cGUACGCGGAAUACUUCGptCGAAGUAUUCCGC AttLB GUACGttLB 27 ptCGAAGUAUUCCGC GUACGttL

FIG. 11: Single-stranded antisense siRNAs mediate gene silencing in HeLacells.

Quantification of lamin A/C knockdown after Western blot analysis. Theblot was stripped after lamin A/C probing and reprobed with vimentinantibody. Quantification was performed using a Lumi-Imager (Roche) andLumiAnalyst software to quantitate the ECL signals (AmershamBiosciences), differences in gel loading were corrected relative tonon-targeted vimentin protein levels. The levels of lamin A/C proteinwere normalized to the non-specific GL2 s1RNA duplex.

FIG. 12. Protein composition of affinity purified RISC.

-   (A) Silver-stained SDS-PAGE gel of affinity-selected    ribonucleoprotein complexes after glycerol gradient (5%-20%)    sedimentation. The arrow indicates the band containing eIF2C1 and    eIF2C2. Molecular size markers are indicated on the left. The    asterisk indicates a fraction for which the protein pellet was lost    after precipitation. (B) Target RNA cleavage assay of the collected    fractions. RISC activity peaked in fraction 7 and 8; bu, buffer.

FIG. 13. Mass spectrometric characterization of eIF2C1 and eIF2C2.

The 100 kDa band was analysed by mass spectrometry. Mass spectrumindicating the peptide peaks corresponding to eIF2C2 (A) and eIF2C1 (B).

-   (C) Alignment of eIF2C2 and eIF2C1 amino-acid sequences indicating    the position of the identified peptides. Sequence differences are    indicated by yellow boxes.

FIG. 14. Predicted amino-acid sequences of the six human Argonauteprotein family members.

FIG. 15. Alignment of the sequences of the six human Argonaute proteinfamily members.

Predicted sequences of human eIF2C1-4, HILI and HIWI have been alignedusing ClustaIX program.

FIG. 16. Predicted cDNA sequences of the six human Argonaute proteinfamily members.

FIG. 17. AU members of the Argonaute family but HIWI are expressed inHeLa cells.

RT-PCR analysis on polyA RNA from HeLa cells. (A) Primers (forward andreverse) used for nested and semi-nested PCR amplification of thedifferent Argonautes and expected length of the PCR products. (B)Agarose gel electrophoresis of the obtained PCR products, confirming theexpected length. Left lanes, 100 by DNA ladder.

EXAMPLE 1. Material and Methods

1.1 siRNA Synthesis and Biotin Conjugation

siRNAs were chemically synthesized using RNA phosphoramidites (Proligo,Hamburg, Germany) and deprotected and gel-purified as describedpreviously. 5′ aminolinkers were introduced by couplingMMT-C6-aminolinker phosphoramidite (Proligo, Hamburg), 3′C7-aminolinkers were introduced by assembling the oligoribonucleotidechain on 3′-aminomodifier (TFA) C7 Icaa control pore glass support(Cherngenes, Mass., USA). The sequences for GL2 luciferase siRNAs wereas described (Elbashir et al., 2001a, supra). If 5′-phosphates were tobe introduced, 50 to 100 nmoles of synthetic siRNAs were treated with T4polynucleotide kinase (300 p1 reaction, 2.5 mM ATP, 70 mM Tris-HCl, pH7.6, 10 mM MgCl₂, 5 mM DTT, 30 U T4 PNK, New England Biolabs, 45 min,37° C.) followed by ethanol precipitation.

3′ Terminal ³²pCp labeling (FIG. 3) was performed in a 30 μl reaction(17 μM siRNA, 0.5 μM ³²pCp (110 TBq/mmol), 15% DMSO, 20 U T4 RNA ligase,NEB, and 1× NEB-supplied reaction buffer) for 1.5 h at 37° C., andgel-purified. One half of the pCp-labeled RNA was dephosphorylated (25μl reaction, 500 U alkaline phosphatase, Roche, and Roche-suppliedbuffer, 30 min, 50° C.), followed by phenol/chloroform extraction andethanol precipitation. Half of this reaction was 5′ phosphorylated (20μl reaction, 2 units T4 polynucleotide kinase, NEB, 10 mM ATP,NEB-supplied buffer, 60 min, 37° C.). A quarter of the initialpCp-labeled siRNA was also 5′ phosphorylated (10 μl reaction, 10 units3′ phosphatase-free T4 polynucleotide kinase, Roche, 10 mM ATP,Roche-supplied buffer, 3 min, 37° C.).

For conjugation to biotin, 20 to 65 nmoles of fully deprotectedaminolinker-modified siRNA were dissolved in 100 μl of 100 mM sodiumborate buffer (pH 8.5) and mixed with a solution of 1 mg of EZ-LinkNHS-PC-LC-Biotin (Pierce, Ill., USA) in 100 μl of anhydrousdimethylformamide. The solution was incubated for 17 h at 25° C. in thedark. Subsequently, siRNAs were precipitated by the addition of 60 μl 2M sodium acetate (pH 6.0) and 1 ml ethanol. The RNA pellet was collectedby centrifugation and biotin-conjugated siRNA was separated fromnon-reacted siRNA on a preparative denaturing 18% acrylamide gel (40 cmlength) in the dark. The RNA bands were visualized by 254 nm UVshadowing and minimized exposure time. The bands were excised, and theRNA was eluted overnight in 0.3 M NaCl at 4° C. and recovered by ethanolprecipitation. siRNA duplexes were formed as previously described(Elbashir et al., Methods 26 (2002), 199-213).

1.2 Preparation of S100 Extracts from HeLa Cells

Cytoplasm from HeLa cells adapted to grow at high density was preparedfollowing the Dignam protocol for isolation of HeLa cell nuclei (Dignamet al., Nucleic Acids Res. 11 (1983), 1475-1489). The cytoplasmicfraction was supplemented with KCl, MgCl₂ and glycerol to finalconcentrations of 100 mM, 2 mM and 10%, respectively. At this stage, theextracts can be stored frozen at −70° C. after quick-freezing in liquidnitrogen without loss of activity. S100 extracts were prepared byultracentrifugation at 31.500 rpm for 60 minutes at 4° C. using aSorvall T-865 rotor. The protein concentration of HeLa S100 extractvaried between 4 to 5 mg/ml as determined by Bradford assay.

1.3 Affinity Purification of RISC with 3′ Biotinylated siRNA Duplexes

For affinity purification of siRNA-associated protein complexes fromHeLa S100 extracts, 10 nM of a 3′ double-biotinylated siRNA duplex wereincubated in 0.2 mM ATP, 0.04 mM GTP, 10 U/ml RNasin, 6 μg/ml creatinekinase, and 5 mM creatine phosphate in 60% S100 extract at 30° C. for 30to 60 min and gentle rotation. Thereafter, 1 ml slurry of ImmobilizedNeutravidin Biotin Binding Protein (Pierce, IL, USA) was added per 50 mlof reaction solution and the incubation was continued for another 60 to120 min at 30° C. with gentle rotation. The Neutravidin beads were thencollected at 2000 rpm for 2 minutes at 4° C. in a Heraeus Megafuge 1.0 Rcentrifuge using a swinging bucket rotor type 2704. Effective capturingof RISC components after affinity selection was confirmed by assayingthe supernatant for residual RISC activity with and withoutsupplementing fresh siRNA duplexes. The collected Neutravidin beads werewashed with 10 volumes of buffer A relative to the bead volume (30 mMHEPES, pH 7.4, 100 mM KCl, 2 mM MgCl₂, 0.5 mM DTT, 10% glycerol)followed by washing with 5 volumes of buffer B (same as buffer A withonly 3% glycerol content). The beads were transferred to a 0.8×4 cmPoly-Prep chromatography column (BioRad; CA, USA) by resuspending in 3volumes of buffer B at 4° C., followed by 10 volumes of washing withbuffer B. Washing of the beads was continued by 10 volumes of buffer Bincreased to 300 mM KCl. The column was then reequilibrated with regularbuffer B. To recover native siRNA-associated complexes, the column wasirradiated in the cold room by placing it at a 2 cm distance surroundedby four 312 nm UV lamps (UV-B tube, 8 W, Herotab, Germany) for 30minutes. To recover the photocleaved siRNP solution, the column wasplaced into a 50 ml Falcon tube and centrifuged at 2000 rpm for 1 minuteat 4° C. using again the 2704 rotor. For full recovery of siRNPs, thebeads were once again resuspended in buffer B followed by a second roundof UV treatment for 15 minutes. Both eluates were pooled and assayed fortarget RNA degradation.

1.4 Target RNA Cleavage Assays

Cap-labeled target RNA of 177 nt was generated as described (Elbashir etal., EMBO J. 20 (2001 c), 6877-6888) except that his-tagged guanylyltransferase was expressed in E. coli from a plasmid generously providedby J. Wilusz and purified to homogeneity. If not otherwise indicated, 5′phosphorylated siRNA or siRNA duplex was pre-incubated in supplementedHeLa S100 extract at 30° C. for 15 min prior to addition of cap-labeledtarget RNA. After addition of all components, final concentrations were100 nM siRNA, 10 nM target RNA, 1 mM ATP, 0.2 mM GTP, 10 U/ml RNasin, 30μg/ml creatine kinase, 25 mM creatine phosphate, 50% S100 extract.Incubation was continued for 2.5 h. siRNA-mediated target RNA cleavagein D. melanogaster embryo lysate was performed as described (Zamore etal., Cell 101 (2000), 25-33). Affinity-purified RISC in buffer B wasassayed for target RNA cleavage without preincubation nor addition ofextra siRNA (10 nM target RNA, 1 mM ATP, 0.2 mM GTP, 10 U/ml RNasin, 30μg/ml creatine kinase, 25 mM creatine phosphate, 50% RISC in buffer B).Cleavage reactions were stopped by the addition of 8 vols of proteinaseK buffer (200 mM Tris-HCl pH 7.5, 25 mM EDTA, 300 mM NaCl, 2% w/v SDS).Proteinase K, dissolved in 50 mM Tris-HCl pH 8.0, 5 mM CaCl₂, 50%glycerol, was added to a final concentration of 0.6 mg/ml and processedas described (Zamore et al. (2000), supra). Samples were s separated on6% sequencing gels.

1.5 Analytical Gel Filtration

UV-eluates in buffer B were fractionated by gel filtration using aSuperdex 200 PC 3.2/30 column (Amersham Biosciences) equilibrated withbuffer A on a SMART system (Amersham Biosciences). Fractionation wasperformed by using a flow rate of 40 μl/minute and collecting 100 μlfractions. Fractions were assayed for specific target RNA cleavage. Sizecalibration was performed using molecular size markers thyroglobulin(669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa)and BSA (66 kDa) (Amersham Biosciences).

1.6 Glycerol Gradient Sedimentation

UV-eluates were layered on top of 4 ml linear 5% to 20% (w/w) glycerolgradient adjusted to 30 mM HEPES, pH 7.4, 100 mM KCl, 2 mM MgCl₂, 0.5 mMDTT. Centrifugation was performed at 35000 rpm for 14.5 h at 4° C. usinga Sorvall SW 60 rotor. Twenty fractions of 0.2 ml volume were removedsequentially from the top and 15 μl aliquots were used to assay fortarget RNA cleavage.

2. Results

2.1 A Human Biochemical System for siRNA Functional Analysis

We were interested in assaying siRNA-mediated target RNA degradation inhuman cell extracts, because siRNAs are powerful reagents to knockdowngene expression in human cells but the action of siRNAs in human cellswas uncertain. To investigate whether siRNAs guide target RNAdegradation in human cells with a similar mechanism to the one observedin D. melanogaster (e.g. Elbashir et al. (2001 b), supra), we preparedsubstrates for targeted mRNA degradation as described previously(Elbashir et al. (2001 c), supra). A 5═-³²P-cap-labeled, 177-nt RNAtranscript, derived from a segment of the firefly luciferase gene, wasincubated in HeLa cell S100 or D. melanogaster embryo extracts with a21-nt siRNA duplex in the presence of an ATP regeneration system (FIG.1A, B). siRNA cleavage assays were performed at 25° C. in D.melanogaster lysate and at 30° C. in HeLa S100 extracts for 2.5 h. Afterdeproteinization using proteinase K, the reaction products wereseparated on a 6% sequencing gel.

Similar to the previous observation in D. melanogaster lysate, weobserved the appearance of a cleavage product in HeLa S100 extract atexactly the same position, thus indicating that the siRNA duplex guidestarget RNA cleavage in the human system with the same specificity andmechanism. The cleavage reaction appeared less efficient when comparedto the D. melanogaster system, but this could be explained by the 5-foldlower total protein concentration of HeLa S100 extracts (25 mg/ml vs. 5mg/ml). Similar to D. melanogaster lysates, siRNA duplexes without 5′phosphate were rapidly 5′ phosphorylated in HeLa S100 extracts (seebelow) and the ability to cleave the target RNA was independent of thepresence of a 5′ phosphate on the synthetic siRNA duplexes.

Comparative analysis of the efficiency of siRNA duplexes of differentlength in D. melanogaster lysate and in transfected mammalian cellsindicated that the differences in silencing efficiencies between 20- to25-nt siRNA duplexes were less pronounced in mammalian cells than in D.melanogaster (Elbashir et al. (2002), supra). Duplexes of 24- and 25-ntsiRNAs were inactive in D. melanogaster lysate, whereas the sameduplexes were quite effective for silencing when introduced bytransfection into HeLa cells. We therefore asked whether siRNA duplexesof 20- to 25-nt are able to reconstitute RISC also with approximatelyequal efficiency. Indeed, we observed no large differences in ourbiochemical assay, and the position of target RNA cleavage was aspredicted according to the cleavage guiding rules established in D.melanogaster lysate (data not shown). Our biochemical results thereforesupport the in vivo observations.

2.2 5′ Modification of the Guide siRNA Inhibits RISC Activity

Modification of siRNAs at their termini is important for developingsiRNA-based affinity purification schemes or for Conjugating reportertags for biophysical measurements. The most common method forintroducing reactive side chains into nucleic acids is by chemicalsynthesis using aminolinker derivatives (Eckstein (1991),Oligonucleotides and analogues, 2nd Ed., Oxford UK, Oxford UniversityPress). After complete deprotection of the oligonucleotide, the primaryamine is typically reacted with the N-hydroxysuccinimidyl ester of thedesired compound. We have introduced 5′ and 3′ aminolinkers with six andseven methylene groups as spacers, respectively. The linker-modifiedsiRNA duplexes were tested for mediating target RNA degradation in HeLaS100 extract (FIG. 2A, B). Modification of the 5′-end of the antisenseguide siRNA abolished target RNA cleavage, while modification of neitherthe sense 5′-end nor of both 3′-ends showed any inhibitory effect. In anidentical experiment using D. melanogaster embryo lysate, we observed asimilar pattern of RISC activity although the duplex carrying the 5′aminolinker-modified antisense siRNA showed some residual activity (datanot shown). Presumably, introduction of additional atoms or the changein terminal phosphate electric charge at the 5′-end of the antisensesiRNA interfered with its ability to function as guide RNA. The criticalfunction of the guide siRNAs 5′ end was previously documented (Elbashiret al. (2001 c), supra).

The ability to modify siRNAs at their 3′-end suggests that siRNAs do notplay a major role for priming dsRNA synthesis and do not act as primersfor degenerative PCR. The fate of a siRNA in HeLa S100 extracts wasfollowed directly by incubation of an internally ³²pCp-radiolabeledsiRNA duplexes. The radiolabeled antisense siRNA strand was alsoprepared with different 5′ and 3′ phosphate modifications (FIG. 3A). Alldescribed combinations of siRNA duplexes were fully competent forRISC-dependent target RNA degradation (data not shown). As previouslyobserved for D. melanogaster lysates (Nykänen et al. (2001), supra),rapid 5′ phosphorylation of siRNA duplexes with free 5′ hydroxyl terminiwas apparent. To our surprise, we noted that a small fraction of the 3′phosphorylated antisense siRNA could be ligated to the opposing 5′hydroxyl of the sense siRNA producing a lower mobility band. Theinter-strand ligation was confirmed by changing the length of theunlabeled sense siRNA, which resulted in the expected mobility changesof the ligation product (data not shown). RNA ligase activity waspreviously observed in HeLa S100 extracts and it is mediated by twoenzymatic activities (e.g. Vicente and Filipowicz, Eur. J. Biochem., 176(1988), 431-439). The 3′ terminal phosphate is first converted to a2′,3′-cyclic phosphate requiring ATP and 3′ terminal phosphate cyclase.Thereafter, the opposing 5′ hydroxyl is ligated to the cyclic phosphateend by an as yet uncharacterized RNA ligase. We chemically synthesizedthe predicted 5′ phosphorylated, 42-nt ligation product and found thatit is unable to mediate target RNA cleavage, presumably because it cannot form activated RISC. The majority of the 3′ phosphorylated duplexessiRNA was gradually dephosphorylated at its 3′ end and emergedchemically similar to naturally generated siRNA. Together, theseobservations indicate that the cell has a mechanism to preserve theintegrity of siRNAs. We were unable to detect a proposed siRNA-primedpolymerization product (FIG. 3B), suggesting that siRNAs do not functionas primers for template-dependent dsRNA synthesis in our system.However, we acknowledge that a proposed RNA-dependent polymeraseactivity may have been inactivated during preparation of our extracts.

2.3 siRNAs Incorporated into RISC do not Compete with a Pool of FreesiRNAs

In order to analyze RISC assembly and stability, we tested whethertarget-unspecific siRNA duplexes were able to compete withtarget-specific siRNA duplexes. When specific and non-specific siRNAduplexes were co-incubated in HeLa S100 extracts, increasingconcentrations of unspecific siRNA duplex competed with the formation oftarget-specific RISC (FIG. 4, left lanes). However, when target-specificsiRNAs were pre-incubated in HeLa S100 extract for 15 min in the absenceof competitor siRNA duplex, the assembled siRNA in the target-specificRISC could no longer be competed with the target-unspecific siRNA duplex(FIG. 4, right lanes). This result suggests that RISC is formed duringthe first 15 minutes of incubation and that siRNAs were irreversiblyassociated with the protein components of RISC during the 2.5 h timewindow of the experiment.

2.4 Purification of Human RISC

After having the 3′ termini of siRNAs defined as the most suitableposition for chemical modification, a photo-cleavable biotin derivativewas conjugated to the 3′ aminolinker-modified siRNAs. A photo-cleavablebiotin derivative was selected because of the advantage of recoveringRISC under non-denaturing conditions after capturing complexes onstreptavidin-coated affinity supports. 3′ Conjugation of biotin to thesense, antisense or to both of the strands did not affect target RNAcleavage when compared to non-biotinylated siRNAs (data not shown).siRNA duplexes with biotin residues on both 3′ ends were therefore usedfor affinity purification (FIG. 5A). The double biotinylated siRNAduplex was incubated in HeLa S100 extracts in the presence of ATP, GTP,creatine phosphate, and creatine kinase for ATP regeneration.Thereafter, streptavidin-conjugated agarose beads were added to capturethe biotinylated siRNA ribonucleoprotein complexes (siRNPs) includingRISC. After extensive washing of the collected beads, the siRNPs werereleased by UV irradiation at 312 nm. The eluate cleaved target RNAsequence-specifically, thus indicating that RISC was recovered in itsnative state from the resin. (FIG. 5B, C, lane UV elu). The flow-throughfrom the affinity selection showed no detectable RISC activityindicating complete binding of RISC by the beads (FIG. 6). The affinityeluate was further analyzed by applying it onto a Superdex 200 gelfiltration column (FIG. 5B) as well as a 5%-20% glycerol gradientultra-centrifugation (FIG. 5C). Individual fractions were collected andassayed for target RNA cleavage without the addition of any furthersiRNA. RISC activity appeared between the molecular size markersaldolase (158 kDa) and BSA (66 kDa) after gel filtration or glycerolgradient centrifugation (FIG. 5B, C). The molecular size of human RISCis therefore estimated to be between 90 and 160 kDa, significantlysmaller than the complex previously analyzed in D. melanogaster lysates(Hammond et al. (2000), supra; Nykänen et al. (2001), supra). The smallsize of RISC suggests that Dicer (210 kDa) is not contained in RISC andthat the formation of RISC from synthetic siRNAs may occur independentlyof Dicer. While these results do not rule out a role for Dicer duringassembly of RISC, they emphasize the absence of Dicer in RISC.

2.5 RISC Contains a Single siRNA Strand and can be Reconstituted UsingSingle-Stranded siRNAs

Two models are currently discussed concerning the siRNA strandcomposition of RISC. The first model suggests that both strands of theinitially added siRNA duplex are physically present in RISC, but in anunwound conformation. The second model proposes that RISC carries only asingle siRNA strand, implying loss of one of the siRNA strands duringassembly. The latter model has been favored based on the analogy tomiRNA precursor processing, where only one 21-nt strand accumulated froma dsRNA hairpin precursor. The molecular basis for the asymmetry of themiRNA precursor processing reaction is not yet understood. BecausesiRNAs have symmetric 2-nt 3′-overhangs it is assumed that siRNAduplexes enter RISC with equal probability for both orientations, thusgiving rise to distinct sense and antisense targeting RISCs.

To address the constitution of siRNAs in RISC, we affinity selected theassembled complexes with siRNA duplexes that were biotinylated at onlyone of the two constituting strands or both (FIG. 6). If both strandswere present together in RISC, the cleavage activity should be affinityselected on Neutravidin independently of the position of the biotinresidue. In contrast, we observed target RNA cleavage from UV eluatesafter streptavidin selection only for siRNA duplexes with biotinconjugated to the antisense strand, but not the sense strand (FIG. 6).RISC activity, assembled on siRNA duplexes with only the sense siRNAbiotinylated, remained in the flow-through. These data suggest that RISCcontains only a single-stranded RNA molecule.

To assess whether single-stranded siRNAs may be able to reconstituteRISC, single-stranded 5′ phosphorylated siRNAs as well as the siRNAduplex were incubated at concentrations between 1 to 100 nM withcap-labeled target RNA in HeLa S100 extract (FIG. 7A). At 100 nMsingle-stranded antisense siRNA, we detected RISC-specific target RNAcleavage, thus confirming that single-stranded siRNAs are present inRISC. At lower concentrations of single-stranded siRNAs, RISC formationremained undetectable while duplex siRNAs were effectively forming RISCeven at 1 nM concentration. Therefore, a specific pathway exists whichconverts double-stranded siRNA into single-stranded siRNA containingRISC. Using D. melanogaster embryo lysate, we were unable to detect RISCactivity from antisense siRNA (FIG. 78), presumably because of the highload of single-strand specific ribonucleases (Elbashir et al. (2001 b),supra). Furthermore, 5′ phosphorylated 20- to 25-nt antisense siRNAswere able to mediate RISC-specific target RNA degradation in HeLa S100extract producing the same target RNA cleavage sites as duplex siRNAs ofthis length (data not shown).

Finally, we tested single-stranded and duplex siRNAs for targeting of anendogenous gene in HeLa cells following our standard protocol previouslyestablished for silencing of lamin A/C. 200 nM concentrations ofsingle-stranded siRNAs with and without 5′ phosphate and 100 nMconcentrations of duplex siRNAs were transfected into HeLa cells. LaminA/C levels were monitored 48 h later using immunofluorescence (FIG. 8A)and quantitative luminescence-based Western blot analysis (FIG. 8B).non-phosphorylated antisense siRNA caused a substantial knockdown oflamin A/C to about 25% of its normal level while 5′ phosphoryled siRNAsreduced the lamin A/C content to less than 5%, similar to the reductionobserved with the lamin A/C 5′ phosphorylated (data not shown) ornon-phosphorylated duplex siRNA (FIG. 8). Sense siRNA and GL2 unspecificsiRNA did not affect lamin A/C levels. The levels of non-targetedvimentin protein were monitored and used for normalizing of the loadingof the lanes of the lamin A/C Western blots.

Gene silencing was also observed with phosphorylated as well asnon-phosphorylated antisense siRNAs ranging in size between 19 to 29 nt.The phosphorylated antisense siRNAs were consistently better performingthan the non-phosphorylated antisense, and their silencing efficiencieswere comparable to that of the conventional duplex siRNA (FIG. 11).

2.6 Protein Composition of RISC

In order to identify the protein components of the RNA-induced silencingcomplex (RISC) in HeLa S100 extract, the specific affinity selectionpreviously outlined was used. UV eluates were fractionated on a 5-20%glycerol gradient, fractions were recovered from the gradient andanalysed for protein composition and target RNA endonucleolyticactivity. Two proteins of approximately 100 kDa were identified by massspectrometry in the peak fraction of the endonucleolytic activity (FIG.12, fractions 7 and 8), corresponding to eIF2C1 and eIF2C2/GERp95 (FIGS.13A and B). These proteins are 82% similar and are both members of theArgonaute family (FIG. 13C). The first evidence that Argonaute proteinsare part of RISC was provided by classical biochemical fractionationstudies using dsRNA-transfected D. melanogaster S2 cells (Hammond etal., 2001, supra). The closest relative to D. melanogaster Argonaute2,D. melanogaster Argonaute1, was recently shown to be required for RNAI(Williams and Rubin, PNAS USA 99 (2002), 6889-6894).

Mass spectrometry analysis also revealed the presence of three peptidesbelonging exclusively to the HILI member of the Argonaute family ofproteins. The sequences of those peptides are: NKQDFMDLSICTR, iscorresponding to positions 17-29 of the protein; TEYVAESFLNCLRR,corresponding to positions 436-449 of the protein, and; YNHDLPARIIVYR,corresponding to positions 591-603 of the protein. This finding suggeststhat the protein HILI may also be part of RISC.

In human, the Argonaute family is composed of 6 members, eIF2C1, eIF2C2,eIF2C3, eIF2C4, HILI and HMI (FIG. 14). The alignment of the sixpredicted amino-acid sequences show a high conservation, in particularbetween the eIF2C members, and HILI and HIWI (FIG. 15). Predicted cDNAsequences encoding the Argonaute proteins are also shown (FIG. 16).

The expression of the human Argonaute proteins was also investigated inHeLa cells by RT-PCR analysis using total and poly (A) selected RNA. Allmembers of the family but HIWI were detected (FIG. 17).

3. Discussion

The development of a human biochemical system for analysis of themechanism of RNAi is important given the recent success of siRNAduplexes for silencing genes expressed in human cultured cells and thepotential for becoming a sequence-specific therapeutic agent.Biochemical systems are useful for defining the individual steps of theRNAi process and for evaluating the constitution and molecularrequirements of the participating macromolecular complexes. For theanalysis of RNAi, several systems were developed, with the D.melanogaster systems being the most comprehensive as they enable toreconstitute dsRNA processing as well as the mRNA targeting. Formammalian systems, reconstitution of the mRNA targeting reaction has notyet been accomplished. Here, we describe the development and applicationof a biochemical system prepared from the cytoplasmic fraction of humanHeLa cells, which is able to reconstitute the human mRNA-targetingRNA-induced silencing complex (RISC). Formation of RISC was accomplishedusing either 5′ phosphorylated or non-phosphorylated siRNA duplexes; aswell as single-stranded antisense siRNAs; non-phosphorylated siRNAduplexes and presumably also single-stranded antisense siRNAs arerapidly 5′ phosphorylated in HeLa cell extracts (FIG. 3).

Biochemical Characterization of siRNA Function

Reconstitution of RISC activity was only observed using cytoplasmic HeLaextracts. HeLa nuclear extracts assayed under the same conditions didnot support siRNA-specific target RNA cleavage, thus suggesting thatRISC components are located predominantly in the cytoplasm (data notshown).

Modifications of the 5′ and 3′ termini of siRNAs were tested in order toassess the importance of the siRNA termini for the targeting step. Itwas found that the 5′ end modification of the guide siRNA was moreinhibitory for target RNA cleavage than 3′ end modification.Introduction of the 3′ biotin affinity tag into the target-complementaryguide siRNA enabled us to affinity select sense-RNA-targeting RISC,whereas 3′ biotinylation of the sense siRNA strand resulted in RISCactivity in the flowthrough. Furthermore, the single RNA strandcomposition of RISC was confirmed by reconstituting thesequence-specific endonuclease complex using 5′-phosphorylatedsingle-stranded guide siRNA. The reconstitution of RISC fromsingle-stranded siRNA was however less effective and required 10- to100-fold higher concentrations compared to duplex siRNA. Reconstitutionof RISC from single-stranded siRNA was undetectable using D.melanogaster embryo lysate, which is most likely explained by the highcontent of 5′ to 3′ exonucleases in embryo lysate.

The size of RISC in HeLa lysate was determined by gel filtration as wellas glycerol gradient ultracentrifugation after streptavidin affinitypurification with 3′ biotinylated siRNA duplexes. Sizes for RISC in D.melanogaster systems have been reported within a range of less than 230to 500 kDa, however size determinations were conducted without havingaffinity purified RISC. Our affinity-purified RISC sediments in a narrowrange between the size makers of 66 and 158 kDa. The differences to thereported sizes for RISC are not species-specific as we observed asimilar size for RISC in D. melanogaster S2 cell cytoplasmic extractsafter affinity purification (data not shown).

It has previously been proposed that siRNAs act as primers for targetRNA-templated dsRNA synthesis (Lipardi et al., Cell 107 (2001), 297-307)although homologs for such RNA-dependent RNA polymerases known toparticipate in gene silencing in other systems are not identified in D.melanogaster or mammalian genomes. Analysis of the fate of siRNAduplexes in the HeLa cell system did not provide evidence for such asiRNA-primed activity (FIG. 3), but indicates that the predominantpathway for siRNA-mediated gene silencing is sequence-specificendonucleolytic target RNA degradation.

Single-Stranded 5′ Phosphorylated Antisense siRNAs as Triggers ofMammalian Gene Silencing

It was previously noted that introduction of sense and antisense RNAs ofseveral hundred nucleotides in length into C. elegans was able tosequence-specifically silence homologous genes (Guo and Kemphues, Cell81 (1995), 611-620). Later, it was suggested that the sense andantisense RNA preparation were contaminated with a small amount ofdsRNA, which was responsible for the silencing effect and is a much morepotent inducer of gene silencing (Fire et al. (1998), supra). It ishowever conceivable that antisense RNA directly contributed toinitiation of silencing. Indeed, most recently it was shown thatantisense RNAs between 22 and 40 nt, but not sense RNAs were able toactivate gene silencing in C. elegans (Tijsterman et al., Science 295(2002), 694-697). The authors, however, favored the hypothesis ofsiRNA-primed dsRNA synthesis.

We have shown that modification of the 3′ ends of antisense siRNA didnot interfere with reconstitution of RISC in the human system. Together,these observations suggest that the driving forces for gene silencing inC. elegans may be predominantly dsRNA synthesis followed by Dicercleavage, while in human and possibly also in D. melanogasterRISC-specific target mRNA degradation predominates.

Targeting of endogenously expressed lamin A/C by transfection of duplexsiRNA into HeLa cells was the first reported example of siRNA-inducedgene silencing. Lamin A/C protein was drastically reduced by a laminA/C-specific siRNA duplex within two days post transfection, whileunspecific siRNA duplexes showed no effect. At the time, transfection ofnon-phosphorylated sense or antisense siRNA did not reveal a substantialeffect on lamin A/C levels, although more recently a minor reductionupon antisense siRNA transfection was noticed when similarconcentrations of antisense siRNA were delivered as described in thisstudy. However, the effect was not interpreted as RISC-specific effect.Assaying 5′-phosphorylated antisense siRNAs revealed a substantialincrease in lamin A/C silencing. Probably, 5′ phosphorylated siRNAs aremore stable or enter RISC more rapidly. Alternatively, the 5′ end oftransfected single-stranded s1RNA may be less rapidly phosphorylated inthe cell in comparison to duplex siRNAs.

Finally, it should be noted that HeLa cells are generally poor innucleases and represent one of the preferred mammalian systems forstudying RNA-processing or transcription reactions in vivo and in vitro.However, it can be expected that 5′ phosphorylated single-strandedantisense siRNAs are suitable to knockdown gene expression in other celltypes or tissues with a different content of nucleases, since chemicalstrategies to improve nuclease resistance of single stranded RNA areavailable. The general silencing ability of various cell types may alsodepend on the relative levels of siRNA/miRNA-free eIF2C1 and eIF2C2proteins capable of associating with exogenously delivered siRNAs.

In summary, single-stranded 5′-phosphorylated antisense siRNAs of 19- to29-nt in size broaden the use of RNA molecules for gene silencingbecause they can enter the mammalian RNAi pathway in vitro as well as invivo through reconstitution of RISC. Human eIF2C1 and/or eIF2C2 seem toplay a critical. role in this process. Considering the feasibility ofmodulating the stability and uptake properties of single-stranded RNAs,5′-phosphorylated single-stranded antisense siRNAs may further expandthe utility of RNAi-based gene silencing technology as tool forfunctional genomics as well as therapeutic applications.

Argonaute proteins are a distinct class of proteins, containing a PAZand Piwi domain (Cerutti et al., 2000, supra) and have been implicatedin many processes previously linked to post-transcriptional silencing,however only limited biochemical information is available.

Human eIF2C2 is the ortholog of rat GERp95, which was identified as acomponent of the Golgi complex or the endoplasmic reticulum andcopurified with intracellular membranes (Cikaluk et al., Mol. Biol. Cell10 (1999), 3357-3722). More recently, HeLa cell eIF2C2 was shown to beassociated with microRNAs and components of the SMN complex, a regulatorof ribonucleoprotein assembly, suggesting that eIF2C2 plays a role inmiRNA precursor processing or miRNA function (Mourelatos et al., Genes &Dev. 16 (2002), 720-728). A more provocative hypothesis is that miRNAsare also in a RISC-like complex, which could potentially mediate targetRNA degradation, if only perfectly matched miRNA target mRNAs existed.Sequence analysis using cloned human and mouse, however, did not revealthe presence of such perfectly complementary sequences in the genomes(Lagos-Quintana et al., Science 294 (2001), 853-858). Therefore, miRNPsmay only function as translational regulators of partially mismatchedtarget mRNAs, probably by recruiting additional factors that preventdissociation from mismatched target mRNAs.

Human eIF2C1 has not been linked to gene silencing previously, but it ismore than 80% similar in sequence to eIF2C2 (Koesters et al., Genomics61 (1999), 210-218). This similarity may indicate functional redundancy,but it is also conceivable that functional RISC may contain eIF2C1 andeIF2C2 heterodimers. The predicted molecular weight of thisheterodimeric complex would be slightly larger than the observed size of90-160 kDa, but because size fractionation is based on globular shape,we can not disregard this possibility at this time.

Due to the high conservation between the members of the Argonautefamily, it is possible that peptides that derive from regions 100%conserved in the 6 predicted proteins, may belong to members others thaneIF2C1 and eIF2C2. In this respect, three peptides were identified withmasses corresponding to HILI, meaning that this protein might be also acomponent of RISC.

To precisely assess the protein composition of RISC, reconstitution ofthe siRNA-mediated target RNA cleavage must be achieved by usingrecombinant proteins which may be obtained by cloning and expression insuitable bacterial or eukaryotic systems.

We expect that the biochemical characterization or the siRNA-mediatedtarget RNA degradation process will have immediate applications, such asthe development of cell lines or transgenic animals overexpressing RISCcomponents. The efficiency in targeting endogenous genes in those linesor organisms will be enhanced. Furthermore, a reconstituted in vitrosystem for RNAi will allow the design of more potent and specific siRNAto achieve gene silencing.

1. Purified human RISC having a molecular weight of from up to about150-160 kDa.
 2. The RISC of claim 1 comprising at least one member ofthe Argonaute family of proteins.
 3. The RISC of claim 1 containingeIF2C1 and/or eIFC2 and optionally at least one of eIFC3, eIFC4, HILIand HIWI.
 4. The RISC of claim 1, further containing an RNA component,particularly a single-stranded RNA molecule.
 5. The RISC of claim 4,wherein the single-stranded RNA molecule has a length from 14-50nucleotides wherein at least the 14-20 5′ most nucleotides aresubstantially complementary to a target transcript.
 6. The RISC of claim4, wherein said RNA molecule has a length from 15-29 nucleotides.
 7. TheRISC of claim 4, wherein said RNA molecule has a free 5′ hydroxyl moietyor a moiety selected from phosphate groups or analogues thereof.
 8. TheRISC of claim 7, wherein said RNA molecule has a 5′-moiety selected from5′-monophosphate ((HO)2(O)P—O-5′), 5′-diphosphate((HO)2(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated or non-methylated)(7m-G-0-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO) (O)—O-5′), 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-monothiophosphate(phosphorothioate; (HO)2(S)P—O-5′), 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).
 9. The RISC of claim 1, wherein said RNA molecule iscompletely complementary to said target transcript, optionally with theexception of nucleotides that extend beyond position 20 (counted fromthe 5′ terminus).
 10. The RISC of claim 1, wherein said RNA moleculecomprises at least one modified nucleotide analogue, which is preferablyselected from sugar-backbone- and nucleobase-modified ribonucleotidesand combinations thereof.
 11. The RISC of claim 1, wherein said RNAmolecule is associated with biodegradable polymers or microparticles,preferably wherein said association comprises a covalent coupling, inparticular a covalent coupling via the 3′-terminus of the RNA molecule.12. A host cell or non-human host organism capable of overexpressingRISC according to claim
 1. 13. A method of enhancing RNAi in a cell oran organism comprising causing said cell or organism to overexpress atleast one component of RISC according to claim
 1. 14. The RISC moleculeaccording to claim 1 for use as a target for diagnosis and/or therapy.15. The RISC according to claim 1 for use as a diagnostic and/ortherapeutic agent itself, as a molecular-biological reagent or ascomponent in a screening procedure for identification and/orcharacterization of pharmaceutical agents.